Tumor-conditioned macrophages secrete migration-stimulating factor: a new marker for M2-polarization, influencing tumor cell motility.

Tumor-associated macrophages (TAMs) are key orchestrators of the tumor microenvironment directly affecting neoplastic cell growth, neoangiogenesis, and extracellular matrix remodeling. In turn, the tumor milieu strongly influences maturation of TAMs and shapes several of their features. To address the early macrophage (M) differentiation phase in a malignant context, we mimicked a tumor microenvironment by in vitro coculturing human blood monocytes with conditioned media from different cancer cell lines. Only 2 out of 16 tumor cell lines induced M differentiation due to secreted M-CSF isoforms, including high molecular mass species. A global gene profiling of tumor-conditioned M was performed. Comparison with other datasets (polarized M1-M, M2-M, and TAMs isolated from human tumors) highlighted the upregulation of several genes also shared by TAM and M2-polarized M. The most expressed genes were selenoprotein 1, osteoactivin, osteopontin, and, interestingly, migration-stimulating factor (MSF), a poorly studied oncofoetal isoform of fibronectin. MSF (present in fetal/cancer epithelial and stromal cells but not in healthy tissues) was never identified in M. MSF production was confirmed by immunohistochemistry in human TAMs. MSF was induced by M-CSF, IL-4, and TGFbeta but not by proinflammatory stimuli. RNA and protein analysis clearly demonstrated that it is specifically associated with the M2 polarization of M. Tumor-conditioned M-derived MSFs strongly stimulated tumor cell migration, thus contributing to the motile phenotype of neoplastic cells. In conclusion, MSF is a new molecule associated with the M2 polarization of M and expressed by TAMs. Its biological function may contribute to M-mediated promotion of cancer cell invasion and metastasis.

Inflammation-mediated promotion of invasion and metastasis.

Inflammation has been suggested to represent the seventh hallmark of cancer. Myelomonocytic cells are a key component of cancer-related inflammation. Tumor-associated macrophages and their mediators affect key elements in the multistep process of invasion and metastasis, from interaction with the extracellular matrix to the construction of a pre-metastatic niche. Evidence indicating that inflammatory mediators affect genetic stability and cause persistent epigenetic alterations suggests that inflammatory components of the tumor microenvironment impacts on fundamental mechanisms responsible for the generation of metastatic variants. These results provide impetus for efforts aimed at translating cancer-related inflammation into diagnostic-prognostic markers and innovative therapeutic strategies.

Secretome analysis of multiple pancreatic cancer cell lines reveals perturbations of key functional networks.

The cancer secretome is a rich repository in which to mine useful information for both cancer biology and clinical oncology. To help understand the mechanisms underlying the progression of pancreatic cancer, we characterized the secretomes of four human pancreatic ductal adenocarcinoma (PDAC) cell lines versus a normal counterpart. To this end, we used a proteomic workflow based on high-confidence protein identification by mass spectrometry, semiquantitation by a label-free approach, and network enrichment analysis by a system biology tool. Functional networks significantly enriched with PDAC-dysregulated proteins included not only expected alterations within key mechanisms known to be relevant for tumor progression (e.g., cell-cell/cell-matrix adhesion, extracellular matrix remodeling, and cytoskeleton rearrangement), but also other extensive, coordinated perturbations never observed in pancreatic cancer. In particular, we highlighted perturbations possibly favoring tumor progression through immune escape (i.e., inhibition of the complement system, deficiency of selected proteasome components within the antigen-presentation machinery, and inhibition of T cell cytoxicity), and a defective protein folding machinery. Among the proteins found concordantly oversecreted in all of our PDAC cell lines, many are reportedly overexpressed in pancreatic cancer (e.g., CD9 and Vimentin), while others (PLOD3, SH3L3, PCBP1, and SFRS1) represent novel PDAC-secreted proteins that may be worth investigating.

The inflammatory micro-environment in tumor progression: the role of tumor-associated macrophages.

The link between inflammation and cancer proposed more than a century ago by Rudolf Virchow, who noticed the infiltration of leukocytes in malignant tissues, has recently found a number of genetic and molecular confirmations. Experimental, clinical and epidemiological studies have revealed that chronic inflammation contributes to cancer progression and even predisposes to different types of cancer. Cancer-associated inflammation includes: the presence of leukocyte infiltration; the expression of cytokines such as tumor necrosis factor (TNF) or interleukin (IL)-1; chemokines such as CCL2 and CXCL8; active tissue remodelling and neo-angiogenesis. Tumor-associated macrophages (TAM) are key regulators of the link between inflammation and cancer. Many observations indicate that, in the tumor micro-environment, TAM have several protumoral functions, including expression of growth factors, matrix proteases, promotion of angiogenesis and suppression of adaptive immunity. In this review we will discuss the role of TAM in the inflammatory micro-environment of solid tumors and will try to identify potential target for future therapeutic approaches.

Tumor-associated macrophages as incessant builders and destroyers of the cancer stroma.

Tumor-Associated Macrophages (TAM) are key components of the reactive stroma of tumors. In most, although not all cancers, their presence is associated with poor patient prognosis. In addition to releasing cytokines and growth factors for tumor and endothelial cells, a distinguished feature of TAM is their high-rate degradation of the extra-cellular matrix. This incessant stroma remodelling favours the release of matrix-bound growth factors and promotes tumor cell motility and invasion. In addition, TAM produce matrix proteins, some of which are typical of the neoplastic tissues. The gene expression profile of TAM isolated from human tumors reveals a matrix-related signature with the up-regulation of genes coding for different matrix proteins, as well as several proteolytic enzymes. Among ECM components are: osteopontin, osteoactivin, collagens and fibronectin, including also a truncated isoform of fibronectin termed migration stimulation factor. In addition to serve as structural proteins, these matrix components have key functions in the regulation of the vessel network, in the inductionof tumor cell motility and degradation of cellular debris. Among proteolytic enzymes are: matrix metalloproteases, cathepsins, lysosomal and ADAM proteases, and the urokinase-type plasminogen activator. The degrading activity of TAM, coupled to the production of bio-active ECM proteins, co-operate to the build-up and maintenance of an inflammatory micro-environment which eventually promotes tumor progression.

Tumor-associated glycoprotein (TAG-72) is a natural ligand for the C-type lectin-like domain that induces anti-inflammatory orientation of early pregnancy decidual CD1a+ dendritic cells.

Tumor-associated glycoprotein-72 (TAG-72) is physiologically present in secretory phase endometrium, but its presence and possible immunological role in early normal human pregnancy decidua has not received attention. The double labeling of paraffin-embedded early pregnancy decidua sections using B-72.4 anti-TAG-72 mAb and MNF 116 anti-cytokeratin mAb revealed the absence of TAG-72 in uterine decidua of normal and pathological pregnancies (non-embryonic pregnancy and missed abortion) at the implantation sites, although it was present in epithelial cells at and away from the tubal implantation site of an ectopic pregnancy. TAG-72 binds and internalizes by reacting with the mannose receptor (MR-CD206) or with DC-specific ICAM reacting non-integrin (DC-SIGN-CD209) on decidual CD1a+ cells. Decidual CD1a+ cells stimulated with TAG-72 decreased CD83 expression and diminished IL-15 and IFN-γ intracellular production. TAG-72-treated CD1a+ cells decreased IFN-γ production in syngenic decidual and allogenic cord blood T cells even in the presence of lipopolysaccharide. TAG-72- and lipopolysaccharide-pre-treated CD1a+ cells significantly increased IL-4 expression in allogenic cord blood T cells. TAG-72 increased allogenic cord blood T cell proliferation, mediated by decidual CD1a+ cells, compared with its effect on the proliferation of syngenic decidual T cells. All these data emphasize the anti-inflammatory properties of TAG-72-treated decidual CD1a+ cells in terms of their interaction with T cells. Thus, the absence of TAG-72 at the maternal-fetal interface during early pregnancy could lead to a mild pro-inflammatory response that may be beneficial for pregnancy success and trophoblast growth control.

Role of CX3CR1/CX3CL1 axis in primary and secondary involvement of the nervous system by cancer.

CX3CL1 or Fractalkine is a peculiar chemokine that can exist either in a soluble form, like all the other chemokines, and as a cell membrane molecule. CX3CL1 is one of the most expressed chemokines in the central nervous system, where it regulates the communication between neurons, glia and microglia. CX3CR1-expressing microglia may have an important role in limiting tissue injury during inflammation and neuro-degeneration. Recent evidence has implicated CX3CL1 and its cognate receptor CX3CR1 in cancer. Tumors of neural origin (glioma, neuroblastoma) express CX3CR1 which is involved in the adhesion, transendothelial migration and mobilization of tumor cells. In addition, tumors of non-neural origin, like prostate, pancreas and breast carcinoma express high levels of the CX3CR1 receptor. As for other chemokine receptors, CX3CR1 expression is associated with increased migration and site specific dissemination. In pancreatic cancer, receptor expression is involved in the perineural invasion and dissemination of neoplastic cells along intra- and extra-pancreatic nerves. This peculiar route of tumor spread is used also by other carcinomas (e.g. prostate, head and neck) and may represent a target for therapeutic intervention.

Human glioblastoma tumours and neural cancer stem cells express the chemokine CX3CL1 and its receptor CX3CR1.

Human gliomas represent an unmet clinical challenge as nearly two-thirds of them are highly malignant lesions with fast progression, resistance to treatment and poor prognosis. The most severe form, the glioblastoma multiforme, is characterised by a marked and diffuse infiltration through the normal brain parenchyma. Given the multiple effects of chemokines on tumour progression, aim of this study was to analyse the expression of the chemokine CX3CL1 and of its specific receptor CX3CR1 in 36 human surgical glioma samples, with different degrees of histological malignancy and in glioblastoma-derived neurospheres. Herein we show that both ligand and receptor are expressed at the mRNA and protein levels in most specimens (31/36). While receptor expression was similarly detected in low or high grade tumours, the uppermost scores of CX3CL1 were found in grades III-IV tumours: oligodendrogliomas, anaplastic astrocytomas and glioblastomas. Accordingly, the expression of CX3CL1 was inversely correlated with patient overall survival (p = 0.01). Glioblastoma-derived neurospheres, containing a mixed population of stem and progenitor cells, were positive for both CX3CR1 and for the membrane-bound chemokine, which was further up-regulated and secreted after TNF-IFNγ stimulation. Confocal microscopy of 3D neurospheres showed that the ligand was primarily expressed in the outer layer cells, with points of co-localisation with CX3CR1, indicating that this ligand-receptor pair may have important intercellular adhesive functions. The high expression of CXC3L1 in the most severe forms of gliomas suggests the involvement of this chemokine and its receptor in the malignant behaviour of these tumours.

Interleukin-17-producing T-helper cells as new potential player mediating graft-versus-host disease in patients undergoing allogeneic stem-cell transplantation.

OBJECTIVES: Graft-versus-host disease (GVHD) is a major obstacle to safe allogeneic hematopoietic stem-cell transplantation, leading to significant mortality. Recently, T-helper (TH)-17 cells have been shown to play a central role in mediating several autoimmune diseases. The aim of our study was to investigate the relationship between TH-17 cells and GVHD occurring in transplanted patients. METHODS: Blood samples were collected from 51 hematopoietic stem-cell transplantation patients and 15 healthy donors. Patients with GVHD were monitored for the presence of TH-17 cells by ELISPOT or flow cytometry in the peripheral blood and by confocal microscopy in GVHD lesions. Cytokine plasma levels were detected by ELISA. RESULTS: An increased TH-17 population (up to 4.8% of peripheral blood CD4+T lymphocytes) was observed in patients with acute GVHD and (up to 2.4%) in patients with active chronic GVHD along with an inflammatory process. In contrast, the percentage of TH-17 cells drastically decreased in patients with inactive chronic GVHD. TH-17 cells consisted of both interleukin (IL)-17+/interferon (IFN)-gamma- and IL-17+/IFN-gamma+ subsets and expressed IL-23 receptor. Interestingly, IFN-gamma+ TH-17 cells were able to infiltrate GVHD lesions as observed in liver and skin sections. Moreover, the proportion of TH-17 was inversely correlated with the proportion of regulatory T cells observed in the peripheral blood and tissues affected by GVHD. Finally, we demonstrated a strong correlation between TH-17 levels and the clinical status of patients with GVHD. CONCLUSIONS: These findings support the hypothesis that TH-17 are involved in the active phases of GVHD and may represent a novel cellular target for developing new strategies for GVHD treatment.

Targeting tumour-associated macrophages.

Clinical and experimental evidence have highlighted that a major leukocyte population present in tumours, the so-called tumour-associated macrophages (TAM), is the principal component of the leukocyte infiltrate supporting tumour growth. Over the years the mechanisms supporting the protumoural functions of TAM have become increasingly clear and in several experimental tumour models, the activation of an inflammatory response (most frequently mediated by macrophages) has been shown to play an essential role for full neoplastic transformation and progression. This evidence strongly supports the idea that TAM are central orchestrators of the inflammatory networks expressed in the tumour microenvironment, and suggest these cells as possible targets of anticancer therapies.